Anal. Chem. 1995,67, 3520-3525
Capillary Electrophoresis/Frontal Analysis for Microanalysis of Enantioseiective Protein Binding of a Basic Drug Toshio Ohara, Akimasa Shibukawa,* and Terumichi Nakagawa Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan
A new HPCE method was developed for the enantioselective determination of the unbound concentration of a basic drug under plasma protein binding equilibrium.The racemic basic drug verapamil (VER) and human serum albumin mixed solution was used as a model sample solution. The sample solution was introduced into a fused-silica capillary hydrodynamically or electrokinetia l l y . During the electrophoresis followinghydrodynamic injection, the unbound drug zone migrated apart from the sample zone and was separated into two zonal peaks with a plateau due to enantiomers by a chiral selector (trimethyl-/kyclodextrin) dissolved in the acidic running buffer solution (PH 2.5). By the electrokinetic introduction of the same sample solution h m the anodic end, oniy the unbound drug entered the capillary and was separated into the enantiomers, which also gave the zonal peaks with plateau. The unbound concentrationof each enantiomer was determined from the plateau peak height. The results obtained by the Merent methods for sample introduction agreed well with those determined by conventional ultrafiltration-chiralHPLC, which was employed as a reference method. The unbound concentrationof (S)-VERwas 1.7 times higher than that of the antipode. The sample size used in the present method was -200 nL, which is about one-thousandth of that in the reference method. The electrokinetic introduction gave a better peak shape than the hydrodynamic introduction. A drug dosed to the body enters the blood stream, where it reaches an equilibrium of binding to plasma proteins such as albumin and al-acid glycoprotein. The protein binding is a reversible and kinetically rapid process. Unbound drug transfers freely to the target organ, whereas it is hard for the bound drug to penetrate the blood vessel wall. Consequently, protein binding of a drug affects significantly the disposition and the exertion of its pharmaceutical effect This is why the quantitative investigation of protein binding is essential to pharmacokinetics and a therapeutic dosing regime~~.l-~ The protein binding of a chiral drug is potentially different between the optical isomers because of the inherent chirality of protein.4~~ Therefore, a stereoselectiveprotein binding study is necessary. (1) Meyer, M. C.; Guttman, D. E. J. Pharm. Sci. 1968,57, 895-918. (2) Vallner, J. J. J. Pham. Sci. 1977,66, 447-465. (3) Kwong, T. C. Clin. Chem. Sci. 1985,151, 193-216. (4) Tucker, G. T.; Lennard, M. S. Phamacol. They. 1989,45, 309-329. (5) Noctor, T. Drug Stereochemisty, 2nd ed.; Wainer, I. W., Ed.; Dekker: New York, 1993; Chapter 12.
3520 Analytical Chemistry, Vol. 67, No. 79,October 7, 7995
So far, equilibrium dialysis and ultrafiltration followed by HPLC analysis have been commonly used for the determination of unbound drug concentration. However, these methods involve adsorption of drug to the membrane, leakage of bound drug through the membrane, and a long time to attain equilibrium. In contrast, the Hummel-Dreyer method and the gel filtration frontal analysis method, based on size exclusion chromatography,6 are free from these problems. Recently, we developed another chromatographic method called high-performancefrontal analysis (HPFA).7-17 The advantages of HPFA over the conventional methods are as follows: (1)Sample solution containiig drug and protein can be directly applied without pretreatment. (2) Errors due to leakage of protein through the membrane and adsorption of drug to the membrane can be avoided. (3) The total drug concentration and the unbound drug concentration in the protein binding equilibrium can be simultaneously determined from the peak area and peak height, respectively, following a single injection.* (4) HPFA can be easily incorporated into an on-line HPLC system. By coupling with a chiral HPLC column, the unbound concentration of a chiral drug can be determined stereoselectively. (5) As low as a few nanomolar or lower unbound drug concentration can be determined with good reprod~cibility.'~-1~ The injection volume required for HPFA becomes smaller as protein binding of the drug becomes stronger.1° For instance, the strong binding of warfarin and human serum albumin (HSA) (the bound drug fraction was -99%) could be analyzed using 40 p L of sample solution injected,g whereas the analysis of carbamazepine and HSA mixed solution (the unbound drug fraction was -30%) required 1.4 mL of sample.8 To reduce the injection volume, we developed a micro-HPFA method where a microbore (6) Korpela, T. IC;Hmanen, J.-P. Aqueous Size-Erclusion Chromatography;Dubin, P. L., Ed.; Elsevier: Amsterdam, 1988 Chapter 13. (7) Shibukawa, A; Nakagawa, T.; Nishimura, N.; Miyake. M.; Tanaka, H. Chem. Pham. Bull. 1989,37,702-706. (8) Shibukawa, A; Nishimura, N.; Nomura, IC;Kuroda, Y.; Nakagawa, T. Chem. P h a m . Bull. 1990,38, 443-447. (9) Shibukawa, A; Nagao, M.; Kuroda, Y.; Nakagawa, T. Anal. Chem. 1990, 62,712-716. (10)Nishimura, N.; Shibukawa, A; Nakagawa, T. Anal. Sci. 1990,6,355-359. (11) Shibukawa, A; Terakita. A; He, J.; Nakagawa, T. J. Pham. Sci. 1992,81, 710-715. (12) Terakita, A;Shibukawa, A; Nakagawa, T. Anal. Sci. 1993,9, 229-232. (13) Shibukawa, A; Nagao, M.; Terakita, A; He, J.; Nakagawa. T. J. Lis. Chromatogr. 1993,16, 903-914. (14) Terakita, A; Shibukawa, A; Nakagawa, T. Anal. Sci. 1994,10, 11-15. (15) Shibukawa, A; Nakao, C.; Sawada, T.; Terakita, A; Morokoshi, N.; Nakagawa, T. J. P h a m . Sci. 1994,83, 868-873. (16) Shibukawa, A; Kadohara, M.; He, J.; Nishimura, M.; Naito, S.; Nakagawa, T. J. Chromatogr. 1995,694, 81-89. (17) Shibukawa, A; Sawada, T.; Nakao, C.; Izumi,T.; Nakagawa, T.J. Chromafogy. 1995,697, 337-343.
0003-2700/95/0367-3520$9.00/0 0 1995 American Chemical Society
HPFA column is by which the injection volume was reduced to one-tenth. Further reduction of the sample size could be achieved by incorporatingfrontal analysis in the HPCE format (HPCE/FA) .18J9 So far, a few HPCE methods have been reported for the estimation of drug-protein binding. Kraak et al. applied the Hummel-Dreyer method, vacancy peak method, and frontal analysis method in HPCE format for the estimation of warfarinbovine serum albumin intera~ti0n.l~In the Hummel-Dreyer method, protein is electrophoresed in the running buffer containing drug, and the bound drug concentration is calculated from the area of the vacant peak. In the vacancy peak method, the capillary is filled with buffer containing drug and protein, and a small plug of buffer is injected. The bound and unbound drug concentrations are calculated from the areas of two negative vacant peaks. However, these methods are reported to be less preferable than the HPCE/FA method.Ig Affinity HPCE has been used for the estimation of the interaction beheen enzyme and cofactors,2O lectin and sugar,21 peptides and d r u g ~ , 2and ~ *protein ~ ~ and Recently, Lloyd et al. applied af6nity HPCE to the analysis of drug-plasma protein binding,25where protein solution is added to a running buffer. The binding constant was calculated from the subsequent change in electrophoretic mobility of the drug. Affinity HPCE allows the simultaneous protein binding assay of plural drugs by injecting the drugs together. This method is practically beneficial to the stereoselective binding study, because the binding constant of each optical isomer can be calculated by using racemates. In addition, the location of binding site on the protein molecule can be ident5ed by using a displacer with a known binding site location. However, the affinity HPCE method has the following problems: (1) Strong W absorption of protein in the running buffer may interrupt the detection of drug. (2) The binding constant is calculated on the basis of a stoichiometrical assump tion. Usually, 1:l binding is supposed. However, the number of binding sites is often more than unity and may be different between the optical isomers. (3) Sometimes the drug peak becomes broadened, which may lead to error in estimation of the binding constant.25 (4) Once protein is adsorbed to the inner surface of capillary, the drug binds to protein not only in the running buffer but also on the inner which may complicate the numerical analysis. In contrast, HPCE/FA can avoid the above mentioned problems. Since drug is separated from protein, the strong UV absorption of the protein does not disturb the analysis. HPCE/ FA followed by Scatchard analysis gives both the binding constant and the number of binding sites.18 The coating of the inner surface of capillary serves to avoid the adsorption of protein.'8 In addition, unlike other HPCE binding assay methods, HPCE/FA (18) Shibukawa, A; Yoshimoto, Y.; Ohara, T.;Nakagawa, T.J. Pharm. Sn'. 1994, 83, 616-619. (19) Kraak, J. C.; Busch, S.; Poppe, H. J. Chromatogr. 1992,608, 257-264. (20)Chu, Y.-H.; Avila, L. 2.; Biebuyck, H. A; Whitesides, G. M. J. Med. Chem. 1992,35, 291552917, (21) Honda, S.; Taga, A; Suzuki, K.; Suzuki, S.; Kakehi, D. J. Chromatogr, 1992, 597,377-382. (22) Carpenter, J. L;Camilleri, P.; Dhanak, D.; Goddall, D. A J. Chem. Soc., Chem. Commun. 1992,804-806. (23) Chu, Y.-H.; Whitesides, G. M. J. 0%.Chem. 1992,57, 3524-3525. (24) Liu, J.; Volk, K. J.; Lee, M. S.; Pucci, M.; Handwerger, S. Anal. Chem. 1994, 66,2412-2416. (25) Lloyd, D.K; Li,S.; Ryan, P. Chirality 1994,6,230-238. (26) Yang, J.; Hage, D. S. Anal. Chem. 1994,66,2719-2725.
allows the determination of the unbound drug concentration. This paper expands the application of the HPCE/FA method to the enantioselective determination of unbound concentrations of a chiral basic drug. Sample was introduced into a fused-silica capillary electrokinetically or hydrodynamically. In HPCE/FA with electrokineticinjection, the principle of frontal analysis effects the selective introduction of the unbound drug into the capillary. In contrast, in HPCE/FA following hydrodynamic injection, frontal analysis occurs inside the capillary to generate the unbound drug zone. EXPERIMENTAL SECTION
Reagents and Materials. Verapamil (VER) hydrochloride and sodium dodecyl sulfate (SDS) were purchased from Wako Pure Chemicals (Osaka, Japan). HSA (globulin and fatty acid free; Catalog No. A-3782) was purchased from S i a (St. Louis, MO) and was used without further purification. Heptakis(2,3,6tri-O methy1);Bcyclodextrin (TM-B-CD;Catalog No. H-4645) was purchased from Sigma. Racemic VER and HSA mixed solutions were made up in sodium phosphate buffer @H 7.4, ionic strength (I) = 0.17) and were shaken gently at 25 "C for 1 h before analysis. Apparatus. A capillary electrophoresis instrument (CUI3000, Otsuka Electronics Co. Ltd., Osaka, Japan) was equipped with a fused-silica capillary (75 pm id.; GL science Inc., Tokyo, Japan) and a UV absorption detector: temperature, 25 "C; detection, 200 nm. After each electrophoretic run, the capillary was sequentially rinsed with 30 mM SDS in phosphate buffer (PH 7.4) and 50 mM sodium phosphate buffer (PH 2.5). Optimization of Running Buffer Condition. Among the three chiral selectors examined (8-CD, DM-P-CD,TM-PCD),TMP-CD was found to give a baseline separation of VER enantiomers in acidic running buffer (PH 2.5) but not in neutral buffer (PH 7.4). TM-b-CD (40 mM) gave a better enantioseparation than 10 or 20 mM TM-P-CD. Thus, the running buffer condition was optimized as 50 mM sodium phosphate buffer (PH 2.5, adjusted by phosphoric acid) containing 40 mM 'I'M-6-CD. Similar HPCE conditionsfor the chiral separation of VER have been r e p ~ r t e d . ~ ~ ~ ~ ~ Electrokinetic Sample Injection. After the capillary (total length 63 cm, effective length 50 cm) was filled with the running buffer, a small volume of neutral buffer (PH 7.4, I = 0.17) was introduced by suction (3 s) into the capillary. Then, the injection end of the capillary was immersed into the sample solution, and a positive voltage (+18 kV) was applied for 30 s. After sample introduction, the injection end of the capillary was transferred to the running buffer, and a positive voltage (+18 kV) was applied to start the electrophoresis. Hydrodynamic Sample Injection. After the capillary (total length 83 cm, effective length 75 cm) was filled with the running buffer, neutral phosphate buffer (PH 7.4, I = 0.17) was introduced into the capillary by suction (15 s for 300 pM VER-550 pM HSA solution or 18 s for 200 pM VER-550 pM HSA solution). Then, the sample solution was introduced hydrodynamically (10 s for 300 pM VER-550 pM HSA solution or 7 s for 200 pM VER-550 pM HSA solution). After sample introduction, the injection end of the capillary was transferred to the neutral phosphate buffer (PH 7.4, I = 0.17), and a positive voltage (+22 kV) was applied to start the electrophoresis. (27) Soini, H.; Riekkola, M.-L.; Novotny, M. V. J. Chromatogr. 1992,608, 265274. (28) Dethy, J.-M.; De Broux, S.; Lesne, M.; Longstreth, J.; Gilbert, P. J. Chromatogr. B 1994,654, 121-127.
Analytical Chemistry, Vol. 67, No. 19, October 1, 1995
3521
electrode capillary
A pH 2.5
c
A
pH 7.4
0 protein
I
I
I
21
22
23
Time(min) pH 7.4
a
chiral selector
bbb
B 1
Figure I. Schematic diagram of the electrokinetic injection.
Calibration Lines. A series of racemic VER standard solutions (50, 100, 200, and 300 pM) dissolved in phosphate buffer (PH 7.4, I = 0.17) were used to prepare calibration lines. In the case of electrokinetic injection, these calibration standards were introduced according to the same procedure as mentioned above. In the case of hydrodynamic injection, the injection times of the neutral buffer and the calibration standards were 5 and 20 s, respectively. The plateau height of each enantiomer was measured in duplicate,and the averaged value was used to make the calibration lines. Good linearity was obtained (r > 0.998). Determination of Unbound VER Concentrations by a UltraiIltration-Chiral HPLC Method. A disposable ultrafiltration kit (Molcut 11, UFPlLGC, Millipore) was used as a reference standard method to determine the unbound concentration of the VER enantiomer. To suppress the adsorption of the drug on the filter membrane, 1 mL of the sample solution was put into the filtration kit and the membrane was saturated with VER by filtration of -200 pL portion of 1 mL sample solution. After discarding the sample solution remaining in the kit, another 1 mL of the same sample solution was applied, and a -200 p L portion of the filtrate containing the unbound drug was obtained. The filtration was performed at 25 "C. A 100 pL portion of the filtrate was subjected to chiral HPLC under the following conditions: Ultron ESOVM column (15 cm x 6 mm i.d., Shinwa Chemical Industries, Kyoto, Japan); mobile phase of 20 mM NaHr Pod-ethanol, 97:3 (v/v); flow rate, 1.2 mL/min; UV detection 275 nm; injection volume, 80 ptL; column temperature, 37 "C. RESULTS
HPCE/FAwithElectrokineticInjection. Figure 1illustrates the electrokinetic sample introduction process. Since electroosmotic flow is not generated under the acidic running buffer condition (PH 2.5), only the unbound drug with a positive net charge is introduced into the capillary from the anodic end, keeping the same R/S ratio as in the sample solution. The unbound drug was then separated into two zones of enantiomers by the chiral selector in the running buffer. The unbound concentration of each enantiomer was calculated from the respective plateau height. In a protein binding study, it is important to investigate the bindability under physiological conditions. Therefore, phosphate buffer of physiological pH (=7.4) was used to prepare the sample solution. However, the optimal pH of the running buffer solution @H 2.5) was far lower than the physiological pH, as mentioned above. Thus, it was feared that direct contact of the sample solution with the running buffer during the sample injection 3522 Analytical Chemistry, Vol. 67, No. 19, October 1 , 1995
(S)-VER
F
I
21
L
I
22
L
I
-
23
Time(min) Figure 2. Electropherograms of (A) 200 pM racemic VER solution and (B) 200 pM racemic VER in 550 p M HSA solution obtained by HPCE/FA with electrokinetic injection. Table 1. Unbound Concentrations of VER Enantiomers Determined by HPCEFA with Electrokinetic Injection and by an Ultrafiltration (UF).Chiral HPLC Method
HPCE/FAb within-runc day-to-dayd UF-chiral HPLCbsc Racemic 300 p M VER-550 pM HSA Cu(R) (uM) C u ( 9 (uM) Cu(S)/Cu(R)
Cu (R) Cu(S) (uM) CuQ/Cu(R)
60.6 (8.92) 60.6 (2.81) 60.9 (4.54) 104 (5.06) 103 (5.06) 100 (3.45) 1.66 (2.29) 1.71 (2.80) 1.70 (3.81) Racemic 200 pM VER-550 pM HSA 44.3 (3.19) 44.5 (1.77) 43.7 (6.78) 74.7 (2.71) 75.2 (9.31) 77.5 (2.01) 1.72 (3.47) 1.75 (1.27) 1.68 (2.33)
Cu(R) and C u ( 9 represent the unbound concentration of (R)-VER and (S)-VER, respectively. Mean and % CV (in parentheses). n = 5. n = 15.
process might disturb the protein binding equilibrium because of the pH difference. To avoid this trouble, a small volume of the neutral phosphate buffer @H 7.4, I = 0.17) was introduced by suction (3 s) prior to sample introduction. After the sample injection, the anodic end of capillary was immersed in the running buffer, and the electrophoresis was carried out. Panels A and B of Figure 2 show the electropherogram of 200 pM racemic VER solution and 200 pM racemic VER-550 pM HSA mixed solution, respectively. VER enantiomers were completely separated by the chiral selector (TM-P-CD). The peak heights in Figure 2A represent the total drug concentration, and those in Figure 2B represent the unbound drug concentration. In Figure 2B, the plateau heights of both peaks are different from each other, indicating enantioselective protein binding. As shown in Table 1, the unbound concentrations of VER enantiomers in 200 or 300 pM racemic VER and 550 pM HSA mixed solution determined by this method agree with those determined by the ultrafiltration-chiral HPLC method. The enantioselectivity in VER-HSA binding can be quantitatively estimated as the enantiomeric ratio of the unbound concentration. The S / R ratio is -1.7, which agrees with the literature reporting that (R)-VER is bound with HSA more tightly than (S)-VERZ9
When a small volume of the neutral buffer was not introduced prior to the sample injection, the S/R ratio of unbound VER concentration in 200 pM VER and 550 pM HSA mixed solution was estimated as 1.4, which is -20% lower than the value (1.7) estimated under neutral conditions (PH7.4). Our preliminary experiment suggested that VER-HSA binding exhibited no enantioselectivity with the acidic buffer. Therefore, the underestimation of the enantioselectivity is ascribable to the disturbance in the protein binding equilibrium caused by contact with the acidic running buffer (PH2.5). The appearance of a plateau region corresponding to the unbound drug concentration indicates that the binding equilibrium near the capillary injection end does not deviate during sample introduction. The decrease in the amount of unbound drug due to the introduction into the capillary can be supplied by the electrophoretic migration of unbound drug from the bulk sample and/or by the sample diffusion. A relatively long-time injection (30 s) was applied in this study to obtain plateau peaks. For HPCE/FA with electrokinetic injection, the apperarance of a plateau region is not essential in principle but convenient in practice. The plateau peak gives us the maximum UV response. Determination of unbound drug concentration from the plateau height is not influenced by the variation in migration time and the error in injection volume, unlike the determination using the peak area or the height of the drug peak without the plateau region. Since a small volume of the neutral buffer preliminarily sucked into the capillary would produce a slight electroosmotic flow, a small volume of the sample solution (total drug protein) may be introduced into the capillary. This leads to overestimation of the unbound drug concentration, if the peak area or the height of the drug peak without a plateau region is used for the determination. In contrast, plateau height is free from this problem, because the introduced sample solution is subjected to frontal analysis to produce the unbound drug zone with the same plateau height as that of the unbound drug zone introduced electrokinetically. After every run, the capillary was washed sequentially with 67 mM phosphate buffer (PH 7.4) containing 30 mM SDS and with 50 mM phosphate buffer (PH 2.5). Washing with SDS solution is indispensable to obtain good reproducibility. Without SDS washing, the unbound concentrations of VER enantiomers in 300 pM racemic VER-550 pM HSA mixed solution were determined as 64.1 iZ 19.9 and 96.4 f 30.4 pM for (R)- and (S)-VER, respectively (n = 5). The % CV was as large as 31.0%and 31.5%, although the mean value was almost the same as that obtained with SDS washing. The % CV was improved to 1.7%and 3.5%, respectively, by the SDS washing. This method was applied to plasma sample. Figure 3 shows the electropherograms of (A) a human plasma blank and 03) 200 pM racemic VER spiked in human plasma. VER enantiomers were separated well from each other and from endogenous plasma components. In the case of electrokinetic injection, the peak response is dependent on the buffer concentration in the sample solution.30Therefore, the unbound drug concentration in plasma cannot be determined precisely. The calibration by using the drug standard dissolved in plasma ultrafiltrate would allow the determination. However, the enantioselectivity in plasma protein
+
(29) Eichelbaum, M.; Somogi, A; von Unruh, G. E.; Dengler, H. J. Eur. J. Clin. P ~ Q ~ w ~ u1981, c o ~ .19,133-137. (30) Huang, X.; Gordon, M. J.; Zare, R N. Anal. Chem. 1988,60,375-377.
Aha/ (AlI I l
0.051
15
15
2o
2o
Time (min)
Tim(min)
Figure 3. Electropherograms of (A) human plasma, (B) 200 pM racemic VER in human plasma obtained by HPCE/FA with electrokinetic injection, and (C) the expanded electropherogram of (B).
binding can be quantitatively analyzed as the peak height ratio between the enantiomers. The SIR radio of unbound VER Concentration in the plasma sample was 1.59 iZ 0.08 (n = 41, which is close to the enantioselectivity in the HSA solution (see Table 1).
HPCE/FA Following Hydrodynamic Injection. After a plug of drug-protein mixed solution (-200 nL) was introduced hydrodynamicallyinto the capillary, positive voltage was applied on the sample injection side. Neither negatively charged protein nor the bound dmg migrated toward this direction, and only the unbound drug with a positive charge migrated toward the cathodic end (detection end), being separated from protein. During this separation process, the drug-protein mixed zone became shorter, and the zone containing only protein became longer. The binding equilibrium in the drug-protein mixed zone remained constant because of the quick release of bound drug from protein. Finally, the whole drug came out of the protein zone and migrated as a zone of the unbound form. The unbound drug zone was then separated into two zones of enantiomers by the chiral selector in the running buffer. The unbound concentration of each enantiomer was calculated from the respective plateau height. Similarly to the above mentioned electrokinetic injection, a small volume of the neutral phosphate buffer (PH7.4, I = 0.17) was introduced by suction prior to the hydrodynamic sample injection. After the sample injection, the anodic end of capillary was immersed in the neutral phosphate buffer (PH 7.4, I = 0.17), and electrophoresis was carried out. Electrophoresis stopped when the anodic end was wrongly put in the acidic running buffer, probably due to the denaturation of the protein by the acidic running buffer. Figure 4A shows the electropherogramof 200 pM racemic VER solution, and Figure 4B shows that of 200 pM racemic VER in 550 pM HSA solution. VER enantiomers were completely separated by the chiral selector (TM-B-CD). The peak height of each enantiomer in Figure 4B was lower than that in Figure 4A because of the protein binding. Figure 4B shows that the plateau heights of both peaks are different from each other. This indicates Analytical Chemistry, Vol. 67,No. 19, October 1, 1995
3523
A
I
I
I
28 Time(min)
27
29
B (S)-VER
A L (R)-VEE I
30
I
31 Time(min)
I
32
Figure 4. Electropherograms of (A) 200 pM racemic VER solution and (B) 200 p M racemic VER in 550 pM HSA solution obtained by HPCUFA following hydrodynamic injection. Table 2. Unbound Concentrations of VER Enantiomers' Determlned by HPCUFA Following Hydrodynamic InJectlon
within-runb,c
day-to-daybld
Racemic 300 pM VER-550 pM HSA Cu(R) OcM) 61.3 (3.59) 65.0 (7.09) cu (3 105 (3.53) 114 (7.04) CU(s)/CU(R) 1.71 (0.65) 1.75 (3.41) Racemic 200 pM VER-550 pM HSA Cu(R) (uM) 38.6 (5.18) 36.9 (7.03) C u ( 9 OLM) CU(s)/CU(R)
69.3 (1.59) 1.80 (3.65)
65.8 (9.48) 1.78 (5.29)
Cu(R)and C u Q represent the unbound concentration of (R)-VER and (a-VER, respectively. Mean and % CV (in parentheses). n = 5. (I
d n = 15.
that the protein binding is enantioselective. Since both enantiomers were introduced in the same amount, the zonal peak of (R)-VER with the lower plateau height became broader than that of (8-VER Table 2 lists the unbound concentration of VER enantiomers determined by this method. These values almost agree with the results determined by HPCE/FA with electrokinetic injection and by the ultraiiltration-HPLCmethod (Table 1). DISCUSSION
In this paper, two chiral HPCE/FA methods with different injection procedure were used for the stereoselective binding assay of a basic drug. The common advantage is the small sample size. The sample volume introduced hydrodynamically was -200 nL. Estimated from the peak area, the sample size for the electrokinetic injection was almost the same as that for the hydrodynamic injection. This sample size is about onethousandth of that for the conventional ultraiiltration method. Although both HPCE/FA methods gave similar results, electrokinetic injection seems to be more convenient than hydrodynamic injection. The HPCE/FA following hydrodynamic injection may confront the overlap of enantiomer peaks, because a lower 3524 Analytical Chemistry, Vol. 67,No. 19,October 1, 1995
unbound drug fraction gives rise to a wider plateau range. In case the unbound fraction is different between enantiomers, the enantiomer with the higher unbound fraction is separated from protein faster than the antipode, and thereafter the binding equilibrium between the protein and the antipode remaining in the sample zone may be varied. Since this change leads to disturbance in the rear part of the plateau, the height should be measured at the front part of the plateau. In contrast, HPCE/FA with electrokinetic injection produces two zonal peaks of enantiomers with similar peak widths regardless of the unbound drug fraction. This is advantageous to analyze a series of samples with a wide range of bound drug fractions under a single separation condition. In addition, electrokinetic injection does not cause a disturbance in the rear part of the plateau. As found from the comparison between Figures 2b and 4b, electrokinetic injection gave better peak shapes than hydrodynamic injection. HPCE/FAwith electrokinetic injection is applicable only when drug and protein migrate the direction opposite to each other. Since most plasma proteins are negatively charged, only a positively charged drug can be applied. On the other hand, HPCE/FA following hydrodynamic injection is, in principle, applicable not only to a basic drug but also to an acidic drug and a neutral drug, as long as we can obtain enough difference in the electrophoretic mobility between drug and protein. Detectability is the common problem in both methods. The S/N of 10pM racemic VER was roughly estimated as 3. However, the therapeutic concentration of VER is much lower (the maximum concentration of VER in human plasma after oral administration of 80 mg of VER is -80 nM). The on-capillary preconcentration using absorbent packed in a capillary is expected to improve the dete~tability.~I Some devices to prolong optical path length using a Zshaped flow cell,32multireflectionflow cell,33and extended light path c a p i l l d 4 are also considered to benefit the detectability. Although these devices increase peak broadening, this will not be a serious problem, because HPCE/FA does not need a very high separation efficiency. The affinity HPCE method and the HPCE/FA method have their own advantages and disadvantages. Affinity HPCE allows the enantioselective estimation of binding constants by using a racemate sample, while HPCE/FA requires a series of samples of each enantiomer to estimate the binding constants based on Scatchard analysis. In contrast, unlike affinity HPCE, HPCE/FA allows the direct determination of unbound concentrations of enantiomers. The enantioselectivity can be quantitatively estimated as the enantiomeric ratio of the unbound drug concentration. Because binding parameters may change depending on the protein ~oncentration,3~,~~ the bindability should be estimated under physiological protein concentrations. If the physiological concentration of HSA (-550 pM) is applied in afsnity HPCE, several problems, such as large UV absorbance, protein adsorption onto the capillary inner wall, high viscosity, and broadening of the drug peak, will interfere with the analysis. In contrast, HPCE/ (31) Swartz, M. E.; Merion, M. J. Chromatoggr. 1993,632,209-213. (32) Chervet, J. P.; van Soest, R E. J.; Ursem, M. J. Chromatogr. 1991,543, 439-449. (33) Wang, T.;Aiken, J. H.; Huie, C. W.; Hartwick, R A. Anal. Chem. 1991,63, 1372-1376. (34) Heiger, D.N. High-pelformoncecapillay electrophoresb, an introduction; Hewlee-Packard: Palo Alto, CA, 1993; pp 100-101. (35) Boobis, S. W.; Chignell, C. F. Biochem. Pharmacol. 1979,28, 751. (36) Igan, Y.; Sugiyama, Y.; Awazu, S.; Hanano. M. J. Pharm. Sci. 1981,70, 1049.
FA is applicable to the sample solution with a high protein concentration. CONCLUSION HPCE/FA with electrokinetic injection and HPCE/FA following hydrodynamic injection allow the simple and quantitative estimation of the enantioselectivity in protein binding of VER, a model basic drug. The unbound concentrations of VER enantiomers in a high concentration of HSA solution can be directly determined. The reliability of these methods was con6rmed by comparison with a conventional ultrdtrationchiral HPLC method.
The chiral HPCE with electrokineticinjection gives a better peak shape than HPCE/FA following hydrodynamic injection. The small sample size (-200 nL) will benefit the stereoselective binding study of plasma proteins, which are precious and dficult to obtain. Received for review April 25, 1995. Accepted July 6, 1995.@ AC9504001 Abstract published in Advance ACS Abstracts, August 15, 1995.
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